Prenatal Diagnosis of Myhre Syndrome in Two Cases: Further Delineation of the Cardiac and External Phenotype.

Myhre syndrome is a rare genetic disease caused by recurrent gain-of-function variants in SMAD4 (Ile500Thr, Ile500Val, Arg496Cys, and Ile500Met) characterized by postnatal short stature with pseudo-muscular build, joint stiffness, variable intellectual disability, hearing loss, and a distinctive pattern of dysmorphic facial features. The course can be severe in some cases, with life-threatening cardiac and pulmonary complications caused by connective tissue involvement. These progressive features over time make early clinical diagnosis difficult but possible by astute clinicians who evaluate young children with autism or short stature and unusual appearance. Only two cases of Myhre syndrome diagnosed during the prenatal period have been reported. Here, we present a detailed description of two unrelated fetuses with Myhre syndrome, each molecularly confirmed by genome or exome sequencing, who underwent fetal examination after termination of pregnancy. One had severe intrauterine growth retardation associated with crossed fused renal ectopia, and the other one had pulmonary atresia with ventricular septal defect (a form of tetralogy of Fallot). Both had mild dysmorphic features with a wide nasofrontal angle. Our results and a systematic prenatal literature review add insight into the early natural history of Myhre syndrome and highlight the contribution of prenatal next-generation sequencing in prenatal diagnosis and the importance of fetal autopsy in Myhre syndrome.

Suppression of NUPR1 in fibroblast-like synoviocytes reduces synovial fibrosis via the Smad3 pathway.

BACKGROUND: Synovial fibrosis is a common complication of knee osteoarthritis (KOA), a pathological process characterized by myofibroblast activation and excessive extracellular matrix (ECM) deposition. Fibroblast-like synoviocytes (FLSs) are implicated in KOA pathogenesis, contributing to synovial fibrosis through diverse mechanisms. Nuclear protein 1 (NUPR1) is a recently identified transcription factor with crucial roles in various fibrotic diseases. However, its molecular determinants in KOA synovial fibrosis remain unknown. This study aims to investigate the role of NUPR1 in KOA synovial fibrosis through in vivo and in vitro experiments. METHODS: We examined NUPR1 expression in the murine synovium and determined the impact of NUPR1 on synovial fibrosis by knockdown models in the destabilization of the medial meniscus (DMM)-induced KOA mouse model. TGF-ß was employed to induce fibrotic response and myofibroblast activation in mouse FLSs, and the role and molecular mechanisms in synovial fibrosis were evaluated under conditions of NUPR1 downexpression. Additionally, the pharmacological effect of NUPR1 inhibitor in synovial fibrosis was assessed using a surgically induced mouse KOA model. RESULTS: We found that NUPR1 expression increased in the murine synovium after DMM surgical operation. The adeno-associated virus (AAV)-NUPR1 shRNA promoted NUPR1 deficiency, attenuating synovial fibrosis, inhibiting synovial hyperplasia, and significantly reducing the expression of pro-fibrotic molecules. Moreover, the lentivirus-mediated NUPR1 deficiency alleviated synoviocyte proliferation and inhibited fibroblast to myofibroblast transition. It also decreased the expression of fibrosis markers α-SMA, COL1A1, CTGF, Vimentin and promoted the activation of the SMAD family member 3 (SMAD3) pathway. Importantly, trifluoperazine (TFP), a NUPR1 inhibitor, attenuated synovial fibrosis in DMM mice. CONCLUSIONS: These findings indicate that NUPR1 is an antifibrotic modulator in KOA, and its effect on anti-synovial fibrosis is partially mediated by SMAD3 signaling. This study reveals a promising target for developing novel antifibrotic treatment.

Jiawei Taohe Chengqi Decoction attenuates CCl4 induced hepatic fibrosis by inhibiting HSCs activation via TGF-ß1/CUGBP1 and IFN-γ/Smad7 pathway.

BACKGROUND: Hepatic fibrosis (HF) is an essential stage in the progression of different chronic liver conditions to cirrhosis and even hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) plays a crucial role in the progression of HF. IFN- γ/Smad7 pathway can inhibit HSCs activation, while TGF-ß1/CUGBP1 pathway can inhibit IFN-γ/Smad7 pathway transduction and promote HSCs activation. Thus, inhibiting the TGF-ß1/CUGBP1 pathway and activating the IFN-γ/Smad7 pathway reverses HSCs activation and inhibits HF. Jiawei Taohe Chengqi Decoction (JTCD) was derived from the Taohe Chengqi Tang in the ancient Chinese medical text titled "Treatise on Febrile Diseases". We found several anti-HF components in JTCD including ginsenoside Rb1 and others, but the specific mechanism of anti-HF in JTCD is not clear. PURPOSE: To elucidate the specific mechanism by which JTCD reverses HF by inhibiting the activation of HSCs, and to establish a scientific foundation for treating HF with Traditional Chinese medicine (TCM). METHODS: We constructed a CCl4-induced mice HF model in vivo and activated human hepatic stellate cell line (LX-2) with TGF-ß1 in vitro, after which they were treated with JTCD and the corresponding inhibitors. We examined the expression of pivotal molecules in the two pathways mentioned above by immunofluorescence staining, Western blotting and RT-PCR. RESULTS: JTCD attenuated liver injury and reduced serum ALT and AST levels in mice. In addition, JTCD attenuated CCl4-induced HF by decreasing the expression of α-SMA, COL1A1 and other markers of HSCs activation in mice liver tissue. Moreover, JTCD effectively suppressed the levels of TGF-ß1, p-Smad3, p-p38MAPK, p-ATF2, and CUGBP1 in vivo and in vitro and upregulated the levels of IFN-γ, p-STAT1, and Smad7. Mechanically, after using the inhibitors of both pathways in vitro, we found that JTCD inhibited the activation of HSCs by restoring the balance of the TGF-ß1/CUGBP1 and IFN-γ/Smad7 pathways. CONCLUSION: We demonstrated that JTCD inhibited HSCs activation and reversed HF by inhibiting the TGF-ß1/CUGBP1 signalling pathway and upregulating the IFN-γ/Smad7 signalling pathway. Moreover, we have identified specific links where JTCD interferes with both pathways to inhibit HSCs activation. JTCD is an effective candidate for the clinical treatment of HF.

Aerosol inhalation of total ginsenosides repairs acute lung injury and inhibits pulmonary fibrosis through SMAD2 signaling-mediated mechanism.

BACKGROUND: Pulmonary fibrosis (PF) is a progressive lung disease caused by previous acute lung injury (ALI), but there is currently no satisfactory therapy available. Aerosol inhalation of medicine is an effective way for treating PF. Total ginsenosides (TG) shows potential for the treatment of ALI and PF, but the effects of inhaled TG remain unclear. PURPOSE: To determine the therapeutic effects of TG in ALI and PF, to assess the superiority of the inhaled form of TG over the routine form, and to clarify the mechanism of action of inhaled TG. METHODS: Ultrahigh-performance liquid chromatography coupled with Q-Exactive Orbitrap mass spectrometry (UPLC-QE-MS) was applied to determine the chemoprofile of TG. A mouse model of ALI and PF was established to evaluate the effects of inhaled TG by using bronchoalveolar lavage fluid (BALF) analysis, histopathological observation, hydroxyproline assay, and immunohistochemical analysis. Primary mouse lung fibroblasts (MLF) and human lung fibroblast cell line (HFL1) were applied to determine the in vitro effects and mechanism of TG by using cell viability assay, quantitative real time PCR (qPCR) assay, and western blot (WB) analysis. RESULTS: The UPLC-QE-MS results revealed the main types of ginsenosides in TG, including Re (14.15 ± 0.42%), Rd (8.42 ± 0.49%), Rg1 (6.22 ± 0.42%), Rb3 (3.28 ± 0.01%), Rb2 (3.09 ± 0.00%), Rc (2.33 ± 0.01%), Rg2 (2.09 ± 0.04%), Rb1 (1.43 ± 0.24%), and Rf (0.13 ± 0.06%). Inhaled TG, at dosages of 10, 20, and 30 mg/kg significantly alleviated both ALI and PF in mice. Analyses of BALF and HE staining revealed that TG modulated the levels of IFN-γ, IL-1ß, and TGF-ß1, reduced inflammatory cell infiltration, and restored the alveolar architecture of the lung tissues. Furthermore, HE and Masson's trichrome staining demonstrated that TG markedly decreased fibroblastic foci and collagen fiber deposition, evidenced by the reduction of blue-stained collagen fibers. Hydroxyproline assay and immunohistochemical analyses indicated that TG significantly decreased hydroxyproline level and down-regulated the expression of Col1a1, Col3a1, and α-sma. The inhaled administration of TG demonstrated enhanced efficacy over the oral route when comparable doses were used. Additionally, inhaled TG showed superior safety and therapeutic profiles compared to pirfenidone, as evidenced by a CCK8 assay, which confirmed that TG concentrations ranging from 20 to 120 µg/ml were non-cytotoxic. qPCR and WB analyses revealed that TG, at concentrations of 25, 50, and 100 µg/ml, significantly suppressed the phosphorylation of smad2 induced by TGF-ß1 and down-regulated the expression of fibrotic genes and proteins, including α-sma, Col1a1, Col3a1, and FN1, suggesting an anti-fibrotic mechanism mediated by the smad2 signaling pathway. In vitro, TG's safety and efficacy were also found to be superior to those of pirfenidone. CONCLUSIONS: This study demonstrates, for the first time, the therapeutic efficacy of inhaled TG in treating ALI and PF. Inhaled TG effectively inhibits inflammation and reduces collagen deposition, with a particular emphasis on its role in modulating the Smad2 signaling pathway, which is implicated in the anti-fibrotic mechanism of TG. The study also highlights the superiority of inhaled TG over the oral route and its favorable safety profile in comparison to pirfenidone, positioning it as an ideal alternative for ALI and PF therapy.

Rutin reduces inflammation and fibrosis via TGF-ß/SMAD pathways in IgA nephropathy induced rats.

AIM: Rutin is a flavonoid glycoside obtained from the plant Ruta graveolens. It was known to have immunosuppressant activities. This study was focused on effect of rutin against immunoglobulin A (IgA) nephropathy. METHODS: IgA nephropathy was induced in Sprague-Dawley rats with various inducing agents described in text. During the later part of the induction phase, rutin was administered. Control group rats did not receive any treatment or inducing agent, induced group rats received only the inducing agents, whereas treatment group received the inducing agents as well as rutin. RESULTS: During the study, various biochemical parameters pertaining to kidney function were evaluated and also, the expression of proteins and cytokines responsible for inflammation and fibrosis were assessed. The effect of rutin in IgA nephropathy was promising as treatment with rutin reduced the deposition of IgA in the glomeruli of rats. Along with this we also tried to establish the probable mechanism of action of rutin and based on the summary of the results it was concluded that rutin reduced the inflammation and fibrosis related to IgA nephropathy by inhibiting the TGF-ß/SMAD pathways and ultimately reducing the expression of α-smooth muscle actin (α-SMA). CONCLUSION: Comprehending all the above consideration, it may be safely said that that rutin alleviated inflammation and also fibrosis mediated by IgA, by suppressing the transforming growth factor-ß (TGF-ß) activities through suppressor of mothers against decapentaplegic pathways and reduced the epithelial-to-mesenchymal transition by downregulating the α-SMA which is a marker for fibrosis.

Induction of Cell Death by Bifidobacterium infantis DS1685 in Colorectal and Breast Cancers via SMAD4/TGF-Beta Activation.

Therapeutic advancements in treatments for cancer, a leading cause of mortality worldwide, have lagged behind the increasing incidence of this disease. There is a growing interest in multifaceted approaches for cancer treatment, such as chemotherapy, targeted therapy, and immunotherapy, but due to their low efficacy and severe side effects, there is a need for the development of new cancer therapies. Recently, the human microbiome, which is comprised of various microorganisms, has emerged as an important research field due to its potential impact on cancer treatment. Among these microorganisms, Bifidobacterium infantis has been shown to significantly improve the efficacy of various anticancer drugs. However, research on the role of B. infantis in cancer treatment remains insufficient. Thus, in this study, we explored the anticancer effect of treatment with B. infantis DS1685 supernatant (BI sup) in colorectal and breast cancer cell lines. Treatment with BI sup induced SMAD4 expression to suppress cell growth in colon and breast cancer cells. Furthermore, a decrease in tumor cohesion was observed through the disruption of the regulation of EMT-related genes by BI sup in 3D spheroid models. Based on these findings, we anticipate that BI sup could play an adjunctive role in cancer therapy, and future cotreatment of BI sup with various anticancer drugs may lead to synergistic effects in cancer treatment.

Analysis of the DNA-binding properties of TGF-ß-activated Smad complexes unveils a possible molecular basis for cellular context-dependent signaling.

Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine that modulates a wide variety of cellular responses by regulating target gene expression. It principally transmits signals via receptor-activated transcription factors Smad2 and Smad3, which form trimeric complexes with Smad4 upon activation and regulate gene expression by binding to genomic DNA. Here, we examined the mechanisms by which TGF-ß regulates the transcription of target genes in a cell context-dependent manner by screening a double-stranded DNA oligonucleotide library for DNA sequences bound to endogenous activated Smad complexes. Screening was performed by cyclic amplification of selected targets (CASTing) using an anti-Smad2/3 antibody and nuclear extracts isolated from three cell lines (A549, HepG2, and HaCaT) stimulated with TGF-ß. The preference of the activated Smad complexes for conventional Smad-binding motifs such as Smad-binding element (SBE) and CAGA motifs was different in HepG2 than in the other two cell lines, which may indicate the distinct composition of the activated Smad complexes. Several transcription factor-binding motifs other than SBE or CAGA, including the Fos/Jun-binding motifs, were detected in the enriched sequences. Reporter assays using sequences containing these transcription factor-binding motifs together with Smad-binding motifs indicated that some of the motifs may be involved in cell type-dependent transcriptional activation by TGF-ß. The results suggest that the CASTing method is useful for elucidating the molecular basis of context-dependent Smad signaling.

Pathological insights into activin A: Molecular underpinnings and therapeutic prospects in various diseases.

Activin A (Act A) is a member of the TGFß (transforming growth factor ß) superfamily. It communicates via the Suppressor of Mothers against Decapentaplegic Homolog (SMAD2/3) proteins which govern processes such as cell proliferation, wound healing, apoptosis, and metabolism. Act A produces its action by attaching to activin receptor type IIA (ActRIIA) or activin receptor type IIB (ActRIIB). Increasing circulating Act A increases ActRII signalling, which on phosphorylation initiates the ALK4 (activin receptor-like kinase 4) type 1 receptor which further turns on the SMAD pathway and hinders cell functioning. Once triggered, this route leads to gene transcription, differentiation, apoptosis, and extracellular matrix (ECM) formation. Act A also governs the immunological and inflammatory responses of the body, as well as cell death. Moreover, Act A levels have been observed to elevate in several disorders like renal fibrosis, CKD, asthma, NAFLD, cardiovascular diseases, cancer, inflammatory conditions etc. Here, we provide an update on the recent studies relevant to the role of Act A in the modulation of various pathological disorders, giving an overview of the biology of Act A and its signalling pathways, and discuss the possibility of incorporating activin-A targeting as a novel therapeutic approach for the control of various disorders. Pathways such as SMAD signaling, in which SMAD moves to the nucleus by making a complex and leads to tissue fibrosis in CKD, STAT3, which drives renal fibroblast activity and the production of ECM, Kidney injury molecule (KIM-1) in the synthesis, deposition of ECM proteins, SERCA2a (sarcoplasmic reticulum Ca2+ ATPase) in cardiac dysfunction, and NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells) in inflammation are involved in Act A signaling, have also been discussed.

Leucine rich α2 glycoprotein 1 derived from malignant pleural mesothelioma cells facilitates macrophage M2 phenotypes.

Background: Macrophages constitute the main part of infiltrating immune cells in Malignant pleural mesothelioma (MPM) and abnormally high ratios of M2 macrophages are present in both pleural effusion and tissue samples of MPM patients. Whether MPM cells affect formation of M2 macrophages is poorly understood. In this study, we focused on identification of MPM-cells-derived soluble factors with M2-promoting effects. Methods: Media of malignant pleural mesothelioma cells were collected and soluble factors affecting macrophages were analyzed by mass spectrometry. TGF-ß receptor inhibitor SB431542 was used as the entry point to explore the downstream mechanism of action by qRT-PCR, WB and immunofluorescence. Results: The serum-free culture media collected from the human MPM cells Meso1 and Meso2 significantly enhanced expression of the M2 signature molecules including IL-10, TGF-ß and CD206 in the human macrophages THP-1, while the culture medium of the human MPM cells H2452 did not show such M2-promoting effects. Analysis of proteins by mass spectrometry and ELISA suggested that Leucine rich α2 glycoprotein 1(LRG1) was a potential candidate. LRG1 time- and dose-dependently increased expression of the M2 signature molecules, confirming its M2-promoting effects. Furthermore, LRG1's M2-promoting effects were reduced by the TGF-ß receptor inhibitor SB431542, and LRG1 increased phosphorylation of Smad2, indicating that M2-promoting effects of LRG1 were via the TGF-ß receptor/Smad2 signaling pathway. Conclusions: Our results provide a potential M2-promoting new member, LRG1, which contributes to the immune escape of MPM via the TGF-ß receptor/Smad2 signaling pathway.

3DMOUSEneST: a volumetric, label-free imaging method evaluating embryo-uterine interaction and decidualization efficacy.

Effective interplay between the uterus and the embryo is essential for pregnancy establishment, however, convenient methods to screen embryo implantation success and maternal uterine response in experimental mouse models are currently lacking. Here we report 3DMOUSEneST, a groundbreaking method for analyzing mouse implantation sites based on label-free higher harmonic generation microscopy, providing unprecedented insights into the embryo-uterine dynamics during early pregnancy. The 3DMOUSEneST method incorporates second-harmonic generation microscopy to image the three-dimensional structure formed by decidual fibrillar collagen, named 'decidual nest', and third-harmonic generation microscopy to evaluate early conceptus (defined as the embryo and extraembryonic tissues) growth. We demonstrate that decidual nest volume is a measurable indicator of decidualization efficacy and correlates with the probability of early pregnancy progression based on a logistic regression analysis using Smad1/5 and Smad2/3 conditional knockout mice with known implantation defects. 3DMOUSEneST has great potential to become a principal method for studying decidual fibrillar collagen and characterizing mouse models associated with early embryonic lethality and fertility issues.

MiR-21 regulates skeletal muscle atrophy and fibrosis by targeting TGF-beta/SMAD7-SMAD2/3 signaling pathway.

Long-term denervation-induced atrophy and fibrosis of skeletal muscle due to denervation leads to poor recovery of muscle function. Studies have shown that the transforming growth factor-ß1 (TGF-ß1)-Smad signaling pathway plays a central role in muscle atrophy and fibrosis. Recent studies demonstrate the role of microRNAs (miRs) in various pathological conditions, including muscle regeneration. miR-21 has been shown to play a dynamic role in inflammatory responses and in accelerating injury responses to fibrosis. We used both RNA sequencing and quantitative RT-PCR strategies to examine the alternations of miRNAs during denervation-induced gastrocnemius muscle atrophy and fibrosis. Our data showed that MiR-21 was upregulated in denervated gastrocnemius muscle tissue, and TGF-ß1treatment increased miR-21 expression. Inhibition of miR-21 reduced gastrocnemius muscle fibrosis and significantly downregulated the expression of p-SMAD2/3 and the fibrosis-associated markers TGF-ß1, connective tissue growth factor, alpha smooth muscle actin. Masson's trichrome staining revealed that atrophy and fibrosis in gastrocnemius muscle tissue were reduced in the miR-21 inhibition group compared to the control group. We confirmed that SMAD7 is a direct target of miR-21 using a dual luciferase assay. Furthermore, Immunofluorescence and Western blot analyses revealed that miR-21 inhibition reduced SMAD2/3 phosphorylation and nuclear translocation. While SMAD7-siRNA abolished the effect. Consequently, the discovery that miR-21 regulates the atrophy and fibrosis of the gastrocnemius muscle offers a possible therapeutic approach for their management.

RHBDF1 modulates cisplatin sensitivity of small cell lung cancer through YAP1/Smad2 signaling pathway.

Small cell lung cancer (SCLC) is a fatal tumor type that is prone to drug resistance. In our previous study, we showed that human rhomboid-5 homolog-1 (RHBDF1) was differentially expressed in 5 intrinsic cisplatin-resistant SCLC tissues compared with 5 intrinsic cisplatin-sensitive SCLC tissues by RNA sequencing, which intrigued us. We performed gain- and loss-of-function experiments to investigate RHBDF1 function, bioinformatics analysis, qRT-PCR, western blotting, and immunoprecipitation to elucidate the molecular mechanisms as well as detect RHBDF1 expression in SCLC by immunohistochemistry. We found that RHBDF1 knockdown promoted cell proliferation and cisplatin chemoresistance and inhibited apoptosis in vitro and in vivo. These effects could be reversed by overexpressing RHBDF1 in vitro. Mechanistically, RHBDF1 interacted with YAP1, which increased the phosphorylation of Smad2 and transported Smad2 to the nucleus. Among clinical specimens, the RHBDF1 was a low expression in SCLC and was associated with clinicopathological features and prognosis. We are the first to reveal that RHBDF1 inhibited cell proliferation and promoted cisplatin sensitivity in SCLC and elucidate a novel mechanism through RHBDF1/YAP1/Smad2 signaling pathway which played a crucial role in cisplatin chemosensitivity. Targeting this pathway can be a promising therapeutic strategy for chemotherapy resistance in SCLC.

Dysregulated Urinary Extracellular Vesicle Small RNAs in Diabetic Nephropathy: Implications for Diagnosis and Therapy.

Background: Diabetic nephropathy (DN) represents a major chronic kidney disorder and a leading cause of end-stage renal disease (ESRD). Small RNAs have been showing great promise as diagnostic markers as well as drug targets. Identifying dysregulated micro RNAs (miRNAs) could help in identifying disease biomarkers and investigation of downstream interactions, shedding light on the molecular pathophysiology of DN. In this study, we analyzed small RNAs within human urinary extracellular vesicles (ECVs) from DN patients using small RNA next-generation sequencing. Method: In this cross-sectional study, urine samples were collected from 88 participants who were divided into 3 groups: type 2 diabetes (T2D) with DN (T2D + DN, n = 20), T2D without DN (T2D - DN, n = 40), and healthy individuals (n = 28). The study focused on isolating urinary ECVs to extract and sequence small RNAs. Differentially expressed small RNAs were identified, and a functional enrichment analysis was conducted. Results: The study revealed a distinct subset of 13 miRNAs and 10 Piwi-interacting RNAs that were significantly dysregulated in urinary ECVs of the DN group when compared to other groups. Notably, miR-151a-3p and miR-182-5p exhibited a unique expression pattern, being downregulated in the T2D - DN group, and upregulated in the T2D + DN group, thus demonstrating their effectiveness in distinguishing patients between the 2 groups. Eight driver genes were identified PTEN, SMAD2, SMAD4, VEGFA, CCND2, CDK6, LIN28B, and CHD1. Conclusion: Our findings contribute valuable insights into the pathogenesis of DN, uncovering novel biomarkers and identifying potential therapeutic targets that may aid in managing and potentially decelerating the progression of the disease.

Isorhamnetin inhibits hypertrophic scar formation through TGF-ß1/Smad and TGF-ß1/CREB3L1 signaling pathways.

Background: Hypertrophic scar (HS) is a common fibrotic skin disease that occurs secondary to burns or injuries. The activation of the TGF-ß signaling pathway contributes immensely to HS formation. Isorhamnetin (ISO) is a type of flavonoid compound that exerts an antifibrotic effect via TGF-ß signaling suppression. However, whether ISO can inhibit HS formation via TGF-ß signaling is yet to be elucidated. This study aimed to examine the influence of ISO on HS pathogenesis and TGF-ß signaling, especially the downstream molecules and networks of TGF-ß signaling that facilitate HS formation. Methods: Hypertrophic scar fibroblasts (HSFBs) were isolated from human HS tissues. The in vitro proliferation, migration, contractile ability, cell cycle, and apoptosis of HSFBs after ISO treatment were determined using cell viability assay, EdU staining, wound healing assay, collagen gel contraction assay, and flow cytometry. The expressions of genes and proteins involved in TGF-ß signaling and its downstream molecules in ISO-treated HSFBs were determined using quantitative PCR (qPCR), immunofluorescence, and western blotting. In vivo, a rabbit HS model was established, and the effects of ISO on rabbit HS formation were investigated using histological analysis, immunohistochemical staining, and qPCR. Results: In vitro studies indicated that ISO treatment suppressed the proliferation, migration, and contractile ability of HSFBs; attenuated the expressions of COL Ⅰ, COL Ⅲ, and α-SMA; and inhibited TGF-ß1 signaling-induced activation of HSFBs by decreasing the levels of phosphorylated Smad2/3 and cleaved CREB3L1 in a dose-dependent manner. Furthermore, ISO augmented apoptosis and G2 phase cell cycle arrest of HSFBs by upregulating the expressions of the proapoptotic proteins Bax and cleaved caspase-3 and downregulating the expression of the antiapoptotic protein Bcl-2. In vivo studies revealed that ISO ameliorated HS formation in the rabbit ear by lowering the scar elevation index, attenuating the collagen density, facilitating the regular arrangement of collagen fibers, and downregulating the expressions of TGF-ß1, CREB3L1, COL Ⅰ, COL Ⅲ, and α-SMA. Conclusions: ISO suppressed HS pathogenesis by dampening TGF-ß1/Smad and TGF-ß1/CREB3L1 signaling pathways, which suggests that it may serve as a candidate inhibitor of TGF-ß1 signaling and a promising anti-HS drug with a high therapeutic potential.

Resveratrol treatment ameliorates hepatic damage via the TGF-ß/SMAD signaling pathway in a phenobarbital/CCl4-induced hepatic fibrosis model.

Objectives: Liver fibrosis is a wound healing response characterized by excessive accumulation of extracellular matrix proteins. This study aimed to investigate the effects of resveratrol treatment on the TGF-ß/SMAD signaling pathway and related biochemical parameters, apoptosis, and liver regeneration phenobarbital-CCl4 induced hepatic fibrosis rat model. Materials and Methods: This model was created through phenobarbital and CCl4 (0.2-0.35 ml/kg). Resveratrol (1 mg/kg/day) was administered to the fibrosis and control groups. Immunohistochemical staining was performed to evaluate αSMA, TGF-ß1, and PCNA in liver tissue. The TUNEL method and Masson's Trichome staining were used to determine apoptosis and collagen accumulation. AST, ALP, ALT, total protein, and total bilirubin levels were measured to determine biochemical status. SMAD2, SMAD3, SMAD4, and SMAD7 expression levels were measured to determine TGF-ß1 related hepatic fibrosis. Results: The SMAD2, SMAD3, and SMAD4 mRNA expression levels were increased and the SMAD7 mRNA expression level was decreased in the fibrosis control group. The SMAD7 mRNA expression level was higher in the phenobarbital-CCl4 induced resveratrol treated group. Increased biochemical parameters indicating hepatic damage, increased number of apoptotic cells, and collagen accumulation surrounding the central vein were observed in the fibrosis group compared with the other groups. It was concluded that administration of resveratrol ameliorates the adverse effects of hepatic fibrosis by regulating biochemical parameters, controlling TGF-ß1/SMAD signaling, enhancing tissue regeneration, and reducing apoptosis in liver cells. Conclusion: Resveratrol can be a beneficial option for the prevention of liver damage in a phenobarbital-CCl4 induced hepatic fibrosis.

Suppression of TGF-ß/Smad3 signaling pathway by Capparis spinosa and quercetin in a rat model of nonalcoholic steatohepatitis.

Objectives: Liver diseases, including non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), pose significant global public health challenges. This study investigates the therapeutic effects of quercetin (QC), Capparis spinosa (CS), a QC and CS combination, and Saroglitazar (SARO) on NASH in a Wistar rat model. Materials and Methods: NASH was induced by a 42-day high-fat diet regimen in male Wistar rats. Post-induction, rats were divided into five groups receiving SARO, QC, CS, and CS+QC combination. We monitored changes in liver and body weights and evaluated the expression of genes associated with fatty acid biosynthesis (e.g., ACC and FAS), ß-oxidation (e.g., CPT1, PPAR α), inflammation (e.g., TNF-α and IL-6), and fibrosis (e.g., TGF-ß and COL1A), as well as protein expression levels of p-Smad2/3 and p-Smad3. Results: Treatment with QC+CS significantly decreased liver weight, body mass gain, and liver triglyceride (TG) compared to other treatments. The QC and CS combined therapy also resulted in a greater normalization of hepatic enzymatic activities, including decreases in ALT and AST levels, coupled with improvements in lipid profile indicated by decreased LDL-C and increased HDL-C concentrations, as compared to SARO and QC alone. Furthermore, this combined treatment significantly down-regulated the expression of TGF-ß, TNF-α, IL-6 genes, and Smad2/3 and Smad3 protein levels. Conclusion: Our study demonstrates that an interactive effect between QC and CS can effectively reduce liver fibrosis and steatosis by inhibiting the TGF-ß/Smad3 signaling pathway in a diet-induced model of nonalcoholic steatohepatitis and fibrosis in rats.

[Modified Fangji Huangqi Decoction alleviates renal interstitial fibrosis by inhibiting TGF-ß1/Smad/Snail signaling pathway during epithelial-mesenchymal transition].

This study investigated the effects of modified Fangji Huangqi Decoction on the expression of proteins related to epithelial-mesenchymal transition(EMT) in a mouse model of unilateral ureteral obstruction( UUO) and in a rat renal tubular epithelial cell(NRK-52E) model of fibrosis induced by transforming growth factor ß1(TGF-ß1). It aims to decipher the molecular mechanism by which modified Fangji Huangqi Decoction alleviates renal interstitial fibrosis. C57/BL mice were subjected to UUO.After the surgery, the mice were treated with 0. 5-fold and 2-fold concentrations of modified Fangji Huangqi Decoction and fosinopril sodium(positive control) for 7 days. The interstitial collagen deposition in the kidney was assessed by Masson staining. Western blot and RT-qPCR were employed to determine the expression levels of TGF-ß1, phosphorylated Smad2/3(p-Smad2/3), Smad2/3, Snail,epithelial cadherin(E-cadherin), alpha smooth muscle actin(α-SMA), and vimentin. The NRK-52E cell model induced by TGF-ß1was treated with the serum samples collected from SD rats treated with different concentrations of modified Fangji Huangqi Decoction.The CCK-8 assay was employed to examine the effects of the serum samples on NRK-52E cell proliferation. The cell morphology in different groups was observed under a microscope. Furthermore, the modeled cells were treated with the serum containing 1-fold decoction. Western blot and RT-qPCR were then employed to measure the expression levels of p-Smad2/3, Smad2/3, Snail,E-cadherin, α-SMA, and vimentin in the cells. Under the same conditions, sh RNA was used to silence the Snail gene, and measurements were repeated before and after treatment with the serum containing 1-fold decoction. The results indicated that modified Fangji Huangqi Decoction alleviated the fibrotic injury in the mouse model of UUO and the fibrosis in the NRK-52E cell model. The treatment with the decoction down-regulated the protein and m RNA levels of EMT-related indicators including p-Smad2/3, α-SMA,Snail, and vimentin, while it up-regulated the expression of E-cadherin. After sh RNA silencing of the Snail gene, the protein and m RNA levels of E-cadherin, α-SMA, and vimentin showed no significant differences before and after treatment with the serum containing the decoction. The results suggest that modified Fangji Huangqi Decoction may alleviate renal interstitial fibrosis by inhibiting the TGF-ß1/Smad/Snail signaling pathway and regulating the EMT process.

Roles of SMAD and SMAD-Associated Signaling Pathways in Nerve Regeneration Following Peripheral Nerve Injury: A Narrative Literature Review.

Although several methods are being applied to treat peripheral nerve injury, a perfect treatment that leads to full functional recovery has not yet been developed. SMAD (Suppressor of Mothers Against Decapentaplegic Homolog) plays a crucial role in nerve regeneration by facilitating the survival and growth of nerve cells following peripheral nerve injury. We conducted a systematic literature review on the role of SMAD in this context. Following peripheral nerve injury, there was an increase in the expression of SMAD1, -2, -4, -5, and -8, while SMAD5, -6, and -7 showed no significant changes; SMAD8 expression was decreased. Specifically, SMAD1 and SMAD4 were found to promote nerve regeneration, whereas SMAD2 and SMAD6 inhibited it. SMAD exerts its effects by promoting neuronal survival and growth through BMP/SMAD1, BMP/SMAD4, and BMP/SMAD7 signaling pathways. Furthermore, it activates nerve regeneration programs via the PI3K/GSK3/SMAD1 pathway, facilitating active regeneration of nerve cells and subsequent functional recovery after peripheral nerve damage. By leveraging these mechanisms of SMAD, novel strategies for treating peripheral nerve damage could potentially be developed. We aim to further elucidate the precise mechanisms of nerve regeneration mediated by SMAD and explore the potential for developing targeted nerve treatments based on these findings.

Decorin attenuates hypertrophic scar fibrosis via TGFß/Smad signalling.

The management of hypertrophic scars (HSs), characterized by excessive collagen production, involves various nonsurgical and surgical interventions. However, the absence of a well-defined molecular mechanism governing hypertrophic scarring has led to less-than-ideal results in clinical antifibrotic treatments. Therefore, our study focused on the role of decorin (DCN) and its regulatory role in the TGF-ß/Smad signalling pathway in the development of HSs. In our research, we observed a decrease in DCN expression within hypertrophic scar tissue and its derived cells (HSFc) compared to that in normal tissue. Then, the inhibitory effect of DCN on collagen synthesis was confirmed in Fc and HSFc via the detection of fibrosis markers such as COL-1 and COL-3 after the overexpression and knockdown of DCN. Moreover, functional assessments revealed that DCN suppresses the proliferation, migration and invasion of HSFc. We discovered that DCN significantly inhibits the TGF-ß1/Smad3 pathway by suppressing TGF-ß1 expression, as well as the formation and phosphorylation of Smad3. This finding suggested that DCN regulates the synthesis of collagen-based extracellular matrix and fibrosis through the TGF-ß1/Smad3 pathway.

SMAD7 Sustains XIAP Expression and Migration of Colorectal Carcinoma Cells.

The reorganization of the cell cytoskeleton and changes in the content of cell adhesion molecules are crucial during the metastatic spread of tumor cells. Colorectal cancer (CRC) cells express high SMAD7, a protein involved in the control of CRC cell growth. In the present study, we evaluated whether SMAD7 regulates the cytoskeleton reorganization and dynamics in CRC. Knockdown of SMAD7 with a specific antisense oligonucleotide (AS) in HCT116 and DLD1, two human CRC cell lines, reduced the migration rate and the content of F-ACTIN filaments. A gene array, real-time PCR, and Western blotting of SMAD7 AS-treated cells showed a marked down-regulation of the X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis family, which has been implicated in cancer cell migration. IL-6 and IL-22, two cytokines that activate STAT3, enhanced XIAP in cancer cells, and such induction was attenuated in SMAD7-deficient cells. Finally, in human CRC, SMAD7 mRNA correlated with XIAP expression. Our data show that SMAD7 positively regulates XIAP expression and migration of CRC cells, and suggest a mechanism by which SMAD7 controls the architecture components of the CRC cell cytoskeleton.